This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The ongoing threat of the use of anthrax as a bioterrorism agent necessitates the development of therapies that block the action of anthrax toxin at any stage of infection. The objective of this research is to understand how pH governs the large conformational change in the protective antigen (PA) to form a membrane spanning pore, a requisite step to initiating the cytotoxicity associated with anthrax, which will guide the development of effective therapeutics directed against anthrax infection. The specific hypothesis is that formation of a pore in the presence of the receptor is critically dependent on the protonation of one or more specific histidine residues. Support for this hypothesis derives from results of preliminary experiments which show that the uniform biosynthetic incorporation of 2-fluorohistidine (2-FHis) (which has a dramatically lower side-chain pKa (pKa ~1)) into the heptamer of PA results in a multimeric protein structure that cannot undergo the pH dependent changes leading to a pore.
Our specific aims are to: 1. Determine the structural basis by which the receptor modulates pore formation. Based on preliminary results, we hypothesize that protonation of histidine residues specifically in the PA receptor binding domain (domain 4) causes a conformational change that results in a loss of binding to the receptor. We plan to isolate and characterize the structure of the receptor binding domain of PA using biophysical methods, including fluorescence, circular dichroism spectroscopy and NMR. 2. Identify specific regions within PA that undergo pH-dependent structural changes that result in pore formation. In addition to the known structural changes that occur in domain 2 of PA, structural changes are likely to occur throughout the protein that are required to facilitate the correct formation of a functional pore. How these structural changes are dependent upon pH will be determined using solution ([13C] and [19F]) and solid-state NMR.
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